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2020

Route Exploration and Synthesis of The Reported Pyridone-Based Route Exploration and Synthesis of The Reported Pyridone-Based

PDI Inhibitor STK076545 PDI Inhibitor STK076545

Eric Greve

Sergey Lindeman

Christina Scartelli

Lin Lin

Robert Flaumenhaft

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Authors Authors Eric Greve, Sergey Lindeman, Christina Scartelli, Lin Lin, Robert Flaumenhaft, and Chris Dockendorff

Marquette University

e-Publications@Marquette

Chemistry Faculty Research and Publications/College of Arts and Sciences

This paper is NOT THE PUBLISHED VERSION; but the author’s final, peer-reviewed manuscript. The published version may be accessed by following the link in the citation below.

Organic & Biomolecular Chemistry, Vol. 18, No. 34 (2020): 6665-6681. DOI. This article is © Royal Society of Chemistry and permission has been granted for this version to appear in e-Publications@Marquette. Royal Society of Chemistry does not grant permission for this article to be further copied/distributed or hosted elsewhere without the express permission from Royal Society of Chemistry.

Route Exploration and Synthesis of The Reported Pyridone-Based PDI Inhibitor STK076545†

Eric Greve Department of Chemistry, Marquette University, Milwaukee, WI Sergey V. Lindeman Department of Chemistry, Marquette University, Milwaukee, WI Christina Scartelli Division of Hemostasis and Thrombosis, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts Lin Lin Division of Hemostasis and Thrombosis, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts Robert Flaumenhaft Division of Hemostasis and Thrombosis, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts

Chris Dockendorff Department of Chemistry, Marquette University, Milwaukee, WI

Abstract The enzyme protein disulfide isomerase (PDI) is essential for the correct folding of proteins and the activation of certain cell surface receptors, and is a promising target for the treatment of cancer and thrombotic conditions. A previous high-throughput screen identified the commercial compound STK076545 as a promising PDI inhibitor. To confirm its activity and support further biological studies, a resynthesis was pursued of the reported β-keto-amide with an N-alkylated pyridone at the α-position. Numerous conventional approaches were complicated by undesired fragmentations or rearrangements. However, a successful 5-step synthetic route was achieved using an aldol reaction with an α-pyridone allyl ester as a key step. An X-ray crystal structure of the final compound confirmed that the reported structure of STK076545 was achieved, however its lack of PDI activity and inconsistent spectral data suggest that the commercial structure was misassigned.

This article is part of the themed collection: Total synthesis in OBC

Introduction Protein disulfide isomerase (PDI) is an enzyme primarily localized in the endoplasmic reticulum that catalyzes the oxidation–reduction and isomerization of disulfide bonds and serves as a necessary chaperone for protein folding.1 In addition, PDI can be released onto the surface of endothelial and platelet cells, where it acts to promote effective coagulation via mechanisms presently under study. For these reasons, PDI inhibitors are of significant interest both for the treatment of cancer2 and the prevention of thrombosis.3 Several animal models of thrombosis have demonstrated that targeting cell surface PDI with antibodies or small molecules blocks both platelet accumulation and fibrin generation.4–8 Previously reported PDI antagonists suffer from poor selectivity, irreversibility, and/or low potency. In an effort to identify novel inhibitors of PDI with more suitable therapeutic properties, a high-throughput screen was performed by Flaumenhaft and co-workers on approximately 5000 bioactive small molecules.6 They identified a class of flavonoids called quercertins, found in high abundance in various fruits and vegetables, that inhibit PDI. From this class, isoquercertin (Fig. 1) was found to decrease D-dimer plasma concentrations, a biomarker for venous thromboembolic disease, by a median of 22% in a phase II clinical trial.9 However, the high dose and highly variable patients responses are drawbacks of isoquercertin.

To seek additional PDI inhibitors, a second high-throughput screen was performed on 348 505 compounds from the Molecular Libraries Small Molecule Repository.10 Two series of PDI inhibitors, represented by bepristats 1a and 2a (Fig. 1) were found to bind to the hydrophobic pocket of the b’ domain.8 A commercial compound called STK076545 (Fig. 1) was also identified from the high-throughput screen to be a reasonably potent hit for the inhibition of PDI, and had an attractive structure for medicinal chemistry studies relative to other hits. The commercial supply of STK076545 was soon depleted, and we were unable to secure additional

quantities, so we immediately endeavored to synthesize it. Its structure proved to be deceptively simple, and this manuscript describes several pitfalls that were encountered prior to its successful synthesis.

Fig. 1 Select inhibitors of PDI.

Methods for preparing β-keto amides have been pursued for more than a century.11 The most obvious approach to β-keto amides is via amide couplings between β-keto acids and amines, which also permits late stage diversification for medicinal chemistry studies (Fig. 2, approach ‘a’). However, this approach may be complicated by the limited stability of the β-keto acid starting materials, which can undergo decarboxylation (step ‘b’). Alternatively, Meldrum's acid can be C-acylated, then aminolysis affords a β-keto amide, but this approach is limited to α-unsubstituted substrates.12 Direct aminolysis of β-keto esters13 or β-keto thioesters14 at high temperature is possible (approach ‘c’), but can be compromised by competing enamine formation. Aminolysis reactions catalyzed with DMAP,15 enzymes,16 or transition metals17,18 have also been reported. Alternatively, addition of a ketone or enamine to an isocyanate have also been reported (approach ‘d’). Cross Claisen-like condensations of esters with amide enolates have been reported (approach ‘e’),19,20 or alternatively an aldol reaction between a pyridone-containing amide and a benzaldehyde (Ar = Ph for STK076545) could be envisaged, followed by alcohol oxidation (step ‘f’).

Fig. 2 Retrosynthetic strategies for accessing the β-keto amide in STK076545 (Ar = Ph, R1 = –CH2CH2NEt2).

Other approaches involving a late stage addition of the pyridone are possible, but these were not initially considered since we were first interested in exploring amide structure–activity relationships (SARs), and the

presence of a basic tertiary amine on the amide side chain of STK076045 could complicate a late stage halogenation/pyridone N-alkylation reaction. It remained to be determined how the presence of an α-pyridone could affect the steps outlined in Fig. 2. Herein, we report our explorations of these routes, culminating in a successful 5-step synthesis of the reported structure of STK076545. An initial version of this work was deposited in ChemRxiv on May 25th, 2020.21

Results and discussion β-Keto carboxylic acid The initial synthetic route we envisioned to access STK076545 (Fig. 2, approach ‘a’) involved N-alkylation of 2-pyridone with bromo-β-keto ester 2, followed by ester hydrolysis and amide coupling (Scheme 1). Using conditions previously reported using a bromomalonate,22 both the N-alkylated 3 and O-alkylated 4 products were isolated, in 47% and 9% yield respectively. 3 and 4 were readily distinguishable based on their 13C NMR chemical shifts for the α-carbon, with the O-alkylated product 4 being assigned based on the more downfield α-carbon peak at 75.9 ppm. In this paper, all alkylations of 2-pyridone gave N-alkylation as the major product, though the O-alkylated products were sometimes observed in trace amounts. Unfortunately, the hydrolysis of ester 3 under acidic (H2SO4) or basic conditions (NaOH, LiOH, or Me3SnOH23) all resulted in decarboxylation of carboxylic acid intermediate 6 to yield ketone 5, despite careful attempted isolations using buffered aqueous media. Direct coupling of alkali metal carboxylate salts has been shown by Batey and coworkers to be a useful strategy with unstable carboxylic acids.24 Attempts at a tandem ester hydrolysis of 3 with NaOH or LiOH followed by peptide coupling with carboxylate 7 were not fruitful. Alternatively, the decarboxylation product 5 was synthesized on a larger scale via N-alkylation of 2-pyridone with 2-bromoacetophenone. Subsequent carboxylation using MgCl2 and NaI with CO2 also gave no detectable amount of carboxylate 8 or carboxylic acid 6 after an acidic workup.25

Scheme 1 Ester hydrolysis and carboxylate formation.

In an effort to access carboxylic acid 6 under milder conditions, the analogous benzyl ester intermediate 13 was synthesized in four steps (Scheme 2). First, 1,3-dicarbonyl 10 was prepared from acetophenone and dimethylcarbonate using NaH in 98% yield.26 ZnO-catalyzed transesterification of 10 afforded benzyl alcohol 11.27 Next, monohalogenation of 11 with NBS catalyzed by Amberlyst-15®, followed by reaction with 2-pyridone yielded α-substituted-β-keto ester 13 in 66% yield over two steps.28 Benzyl removal from ester 13via palladium-catalyzed hydrogenation also resulted in decarboxylation. In addition, 1H NMR analysis of the crude product indicated the ketone was reduced to afford benzyl alcohol 14. Efforts to reduce the ketone prior to ester hydrolysis were not successful (Scheme 7).

Scheme 2 Synthesis and deprotection of benzyl ester 13.

Enolate formation from ketone 5 Instead of proceeding through a carboxylic acid intermediate, we envisioned installation of the amide via reaction of an enolate with a suitable isocyanate, or reaction with CDI followed by addition of an amine to the intermediate acylimidazole (Fig. 2, approach ‘d’). To identify suitable conditions for enolate formation with ketone 5, LDA, LiHMDS, and NaH were screened as bases (Table 1). Reactions were quenched at −78 °C or 20 °C using D2O, and crude samples were analyzed via1H NMR. Based on our screen, it was found that all reactions occurring at 20 °C facilitated enolate formation (entries 3–5), while no deuterium incorporation occurred when quenching the samples at −78 °C (entries 1 and 2). The observed deuterium incorporation over 100% is expected to be within the systematic error associated with the preparation of LDA or the weight of NaH, and 1H NMR integrations of these small scale reactions.

Table 1 Enolate formation from ketone 5

Entry Base Base (eq.)

T (°C) addition of base

T (°C) addition of D2O

% deuterium incorporationa

1b LDA 1.05 −78 −78 0 2b LiHMDS 1.05 −78 −78 0 3b LDA 1.05 −78 20 115 4b LiHMDS 1.05 −78 20 94 5c NaH 1.2 20 20 134

a Deuterium incorporation was determined via1H NMR. b 12.5 mg of 5 was used in this reaction. c 25 mg of 5 was used in this reaction.

The enolate from ketone 5 was next formed using LDA at 20 °C and reacted with CDI or urea intermediate 16, synthesized from CDI and N,N-diethylethylenediamine (Scheme 3). There was no observable reaction with either electrophile, even after heating at 70 °C. We then tested commercially available tert-butyl isocyanate as a model isocyanate for reaction screening. When LDA was used as a base, no desired product was observed. Rather, urea byproduct 18 formed from the addition of diisopropylamine to tert-butyl isocyanate was detected via LC-MS. Switching to NaH as the base and heating the reaction at 100 °C for 2 h in toluene yielded amide 19 in 15% yield. Efforts to synthesize the desired isocyanate from N,N-diethylethylenediamine and triphosgene, or reacting diethylamine with 2-bromoethyl isocyanate, were both troublesome. With this synthetic route being low yielding and having the limitation of only producing secondary amides, we chose to seek an alternative route.

Scheme 3 Enolate reactions with ketone 5.

Direct aminolysis of β-keto ester Another common synthetic approach to access amides is via direct aminolysis of esters (Fig. 2, approach ‘c’). Starting from β-keto ester 3, we first tested a Ag(I)-catalyzed aminolysis (Scheme 4).18 Rather than observing conversion to the desired β-keto amide, reaction monitoring via LC-MS when using condition A showed masses associated with ester 21 and amide 24 (Scheme 5). Similarly, when heating ester 3 with N,N-diethylethylenediamine in toluene at 80 °C with or without DMAP, the same decomposition peaks were present. Ester 21 was isolated when using conditions B and correlated with the mass peak observed via LC-MS. Ag(I)/DBU and DMAP were found to both accelerate the conversion to 21 in a few hours, in comparison to the reaction heated in toluene that proceeded slowly over 24 h. We hypothesize that the β-keto ester decomposes via a retro Claisen-like condensation mechanism. The amine (or nucleophilic catalyst) could add to the ketone, followed by collapse of the tetrahedral intermediate 23 and cleavage of the C–C bond to produce ester 21 (Scheme 5).

Scheme 4 Direct aminolysis attempts with β-keto esters.

Scheme 5 Proposed retro Claisen-like condensation decomposition.

Štefane and Polanc reported a method to prepare β-keto amides from β-keto esters that proceeds via a 1,3,2-dioxaborinane intermediate.29 Reacting β-keto ester 3 with boron trifluoride etherate afforded the boron complex 20b in 82% yield (Scheme 4). Unfortunately, subsequent treatment of 20b with N,N-diethylethylenediamine also resulted in decomposition to ester 21 after only 1 h, and complete decomposition after 24 h. Interestingly, when starting from β-keto ester 10 which does not have the pyridone substituent in the α position, the preparation of the boron complex 20a and treatment with N,N-diethylethylenediamine cleanly afforded β-keto amide 22.

Late stage C2–C3 coupling route Inspired by our observed retro Claisen-like reaction that occurred with a β-keto ester substrate, we examined the feasibility of performing an aldol addition, Claisen-like condensation, or acylation with intermediates 27a-b (Scheme 6). Thus, installation of the C2–C3 bond would occur after amide formation and minimize the possibility of decarboxylation. Synthesis of 27a-b began with N-alkylation of 2-pyridone with ethyl bromoacetate followed by ester hydrolysis, affording the previously reported carboxylic acid 25.30 Amide coupling with either N,N-diethylethylenediamine 26a or PMB-protected amine 26b yielded amides 27a-b. We prepared the PMB-protected amide 26b in an effort to avoid competitive deprotonation of the amide proton during enolization reactions. Extensive efforts to react these pronucleophiles with different bases (NaOMe, NaH, and LDA), electrophiles (aldehyde, ester, or acid chlorides 28a-c), and at variable temperatures (0–160 °C) were all unfruitful.

Scheme 6 C2–C3 coupling attempts on amide 26a–b.

Identification of the aldol adduct as a possible intermediate provided inspiration to access alcohol 32, which would not undergo decarboxylation during ester hydrolysis (Scheme 7). Starting from benzyl esters 12 or 13, we attempted to either protect or reduce the benzylic ketone. Efforts to protect the ketone using ethylene glycol and catalytic p-TsOH with triethyl orthoformate and 4 Å mol sieves or a Dean–Stark trap resulted in no conversion. Alternatively, an attempt to reduce the ketone with DIBAL-H resulted in pyridone reduction, as suggested by the crude 1H NMR spectrum. Switching to NaBH4 produced benzyl ester 33, which we presume proceeds via a similar retro-aldol reaction as observed previously.

Scheme 7 Attempted protection and reduction reactions of benzylic ketone 12 and 13.

Protected benzylic alcohol route To access benzylic alcohols that could be converted to ketones late in the synthesis, we prepared bromohydrins 35a-b from the respective methyl and benzyl cinnamates using NBS and I2 as a catalyst (Scheme 8).31 This also installed alpha halides for pyridone alkylations. Under standard 2-pyridone alkylation conditions, epoxide 36 was exclusively formed. To circumvent this issue, the TBS- or MOM-protected bromohydrins 37a-b were synthesized. It was found that the use of 2,6-lutidine as base was critical, as alternative bases such as DIPEA favored epoxidation over alcohol protection. However, subjecting the TBS- or MOM-protected bromohydrin to pyridone alkylation conditions afforded only alkene 38 in <20% yield. Alternatively, the acetyl-protected alcohol 39 was synthesized using acetic anhydride and catalytic DMAP. N-Alkylation of 2-pyridone using 39 afforded what was initially presumed to be the desired product 40a and alkene 41a in 18% and 9% yield respectively (Scheme 9). Due to the low yields, we screened alternative solvents (acetone) and bases (Cs2CO3) in an effort to increase the yield and selectivity; however, the yield of 40a was not improved.

Scheme 8 Preparation of bromohydrin intermediates.

Scheme 9 N-Alkylation of 2-pyridone with acetyl-protected bromohydrin.

Moving forward with the synthesis, methyl ester and acetate hydrolysis proceeded smoothly to yield the presumed carboxylic acid 42a (Scheme 9). At that time, we did not suspect any issues and completed the synthetic sequence to yield 50, which was initially thought to be STK076545 (Scheme 10). N,N-Diethylethylenediamine was used directly for the peptide coupling to prepare 45; however, the subsequent alcohol oxidation was unsuccessful when using DMP, PDC, IBX, or Bobbitt's salt under basic (2,6-lutidine) or acidic (silica gel) conditions.32 Instead, ethanolamine was TBS-protected to afford 44 and then used in an amide coupling using HATU with carboxylic acid 42 to yield 46 in 91% yield. DMP oxidation of the alcohol proceeded smoothly to afford ketone 47, followed by TBS removal using HCl. In a one-pot reaction, alcohol 48 underwent a mesylation followed by a substitution with diethylamine. The final product was treated with HCl to furnish the HCl salt 50 in 28% yield over 2 steps.

Scheme 10 Synthesis of the incorrect regioisomer of STK076545.

However, analogs 48 and 50 were both found to be inactive in a PDI activity assay measuring the reduction of insulin (cleavage of its disulfide bonds). Obtained X-ray crystal structures of 41b and 42b revealed that the pyridone was in the benzylic position (Scheme 9). A distinct difference between 42a and the X-ray structure of 42b is the relative stereochemistry between the hydroxyl and pyridone substituents. We propose that cyclic acetoxonium ion intermediate 43 is formed, similar to that proposed for the Prévost33 and Woodward34 dihydroxylation reactions, and 2-pyridone then attacks the benzylic carbon. Since we started with (E)-methyl cinnamate, the anti addition of water to the intermediate rac-bromonium ion results in a racemic mixture of bromohydrins 39. Formation of the proposed acetoxonium ion intermediate 43 and subsequent pyridone alkyation would result in inversion of both stereocenters and generate 40b. Ester hydrolysis of 40b then yielded acid 42b, with its relative stereochemistry confirmed by the X-ray crystal structure (Scheme 9).

Aldol addition route To circumvent the unexpected rearrangement through the acetoxonium ion intermediate, we planned to perform the N-alkylation prior to installation of the alcohol/ketone functionality. Inspired by Easton and co-workers’ use of NBS and AgNO3 to generate hydroxy-α-amino acid derivatives,35 we sought to access a benzylic bromide intermediate from 51 (Scheme 11). Ester 21 was reacted with LiHMDS to generate an enolate, followed by alkylation with benzyl bromide to afford 51. Subsequent treatment with NBS and AIBN in MeCN yielded no bromination at the benzylic position. The m/z peak observed in the LC-MS trace confirmed the presence of a brominated product, but the crude 1H NMR spectrum suggested that bromination occurred on the pyridone. Alternatively, benzaldehyde was used instead of benzyl bromide to react with the enolate generated from ester 21 to access the benzylic alcohol directly (Scheme 12). Using identical conditions (LiHMDS and 0 °C), the elimination product 53 was generated. Hydrolysis of ester 53 yielded carboxylic acid 54, which X-ray crystallography confirmed to be the Z alkene.

Scheme 11 Alkylation and attempted benzylic bromination.

Scheme 12 Aldol reactions with α-pyridone ester 21.

With LDA being more commonly used in the literature for aldol addition reactions with esters, we switched our base to LDA (Scheme 12). Quenching the reaction at −78 °C was found to be critical to avoid generation of the elimination product and give the desired alcohol 55. Unfortunately, ester hydrolysis with LiOH yielded the elimination product 53 again. In an effort to avoid generation of this undesired alkene, we synthesized the TBS-protected alcohol 56. This route was unfruitful as ester hydrolysis or cleavage using LiOH, Me3SnOH, or LiI all resulted in the generation of 53.

In order to access the carboxylic acid under milder conditions, we instead synthesized allyl ester 57 (Scheme 13). First, 2-pyridone was alkylated with allyl chloroacetate to yield allyl ester 57, and subsequent aldol addition using benzaldehyde afforded alcohol 58. By increasing the amount of benzaldehyde from 1 to 2 equivalents, we were able to nearly double the yield to 82% for this reaction. Allyl removal with Pd(PPh3)4 produced carboxylic acid 59. Amide coupling and subsequent DMP oxidation of alcohol 60 successfully produced the final β-keto-amide product 61 with the reported structure of STK076545. Incomplete conversion and uncharacterized byproducts were observed with the low yielding DMP oxidation. Alternative oxidation conditions (e.g. PDC, MnO2, Bobbitt's salt,32 and Cu catalyzed aerobic oxidation36) were all unsuccessful. We obtained an X-ray crystal structure corroborating the structure of 61 (Fig. 3). Alcohol analog 60, the final compound 61, and its regioisomeric analog 50 were tested in an assay measuring PDI-catalyzed insulin reduction and aggregation. All compounds were found to be inactive, with rutin used as a positive control (Fig. 4, see Experimental section for details). Interestingly, the 1H NMR spectrum of 61 does not match that of the STK076545 received from the commercial supplier (Table 2). This confirms that the structure of the active PDI-inhibiting compound was misassigned.

Scheme 13 Successful allyl ester route to STK076545.

Fig. 3 X-ray structure of amide 61.

Fig. 4 PDI-mediated insulin aggregation assay.

Table 2 1H NMR chemical shifts of STK076545 and 61

Entry Compound 1H NMR (CDCl3) δ (integration) 1 STK076545 8.83 (1H), 8.05 (2H), 7.46 (1H), 7.39 (2H), 7.31 (1H), 7.25 (1H), 6.55 (1H), 6.15 (1H), 4.57

(2H), 3.57 (2H), 2.93 (6H), 2.62 (1H), 1.21 (6H) 2 61 8.01 (2H), 7.65 (1H), 7.60–7.56 (1H), 7.48–7.44 (1H), 7.40–7.34 (2H), 6.60 (1H), 6.24

(1H), 3.39–3.28 (2H), 2.57–2.48 (6H), 0.96 (6H)

Conclusion Several conventional methods for forming β-keto amides resulted in fragmentation of pyridone-containing intermediates, such as retro-Claisen-like and retro-aldol reactions. Efforts to instead proceed via an acetyl protected bromohydrin resulted in a rearrangement that we propose proceeds via an acetoxonium ion intermediate. Alternatively, we successfully synthesized the reported structure of STK076545 in 5% overall yield (unoptimized) via a 5-step synthetic route proceeding through allyl ester 58. This strategy should prove to be broadly useful in accessing β-keto amides, particularly with an electron-withdrawing α-substituent such as N-pyridone. Unexpectedly, neither the final compound (61) nor several of its analogs were found to inhibit protein disulfide isomerase, and its 1H NMR spectrum does not match that of the active commercial compound STK076545. It is not uncommon for complex natural products to have misassigned structures which require correction after more detailed synthetic and spectroscopic studies.37 However, it is often taken for granted that simpler commercial small molecules are provided in high purity and with structures as advertised, which is not

always the case.38 Our results here highlight the importance of resynthesis and structure validation of active compounds prior to embarking on medicinal chemistry campaigns. Current efforts in our lab are ongoing to elucidate the correct structure of the commercially supplied compound that may have useful PDI inhibitory activity.

Experimental section General information All reagents and solvents were purchased from commercial vendors and used as received, except for diethylamine, which was distilled and stored over 4 Å mol sieves prior to use. Deionized water was purified by charcoal filtration and used for reaction workups and in reactions with water. NMR spectra were recorded on Varian 300 MHz or 400 MHz spectrometers as indicated. Proton and carbon chemical shifts are reported in parts per million (ppm; δ) relative to tetramethylsilane, CDCl3, or CD3OD (1H δ 0, 13C δ 77.16, or 1H δ 3.31, 13C δ 49.0, respectively). NMR data are reported as follows: chemical shifts, multiplicity (br = broad, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet); coupling constant(s) in Hz; integration. Unless otherwise indicated, NMR data were collected at 25 °C. NMR data were processed using MestReNova software. Flash chromatography was performed using Biotage SNAP cartridges filled with 40–60 μm silica gel, or C18 reverse phase columns (Biotage® SNAP Ultra 18) on Biotage Isolera systems, with photodiode array UV detectors. Analytical thin layer chromatography (TLC) was performed on Agela Technologies glass plates with 0.25 mm silica gel with F254 indicator. Visualization was accomplished with UV light (254 nm) and aqueous potassium permanganate (KMnO4) stain followed by heating, unless otherwise noted. Tandem liquid chromatography/mass spectrometry (LC-MS) was performed on a Shimadzu LCMS-2020 with autosampler, photodiode array detector, and single-quadrupole MS with ESI and APCI dual ionization, using a Peak Scientific nitrogen generator. Unless otherwise noted, a standard LC-MS method was used to analyze reactions and reaction products: Phenomenex Gemini C18 column (100 × 4.6 mm, 3 μm particle size, 110 A pore size); column temperature 40 °C; 5 μL of sample in MeOH or CH3CN at a nominal concentration of 1 mg mL−1 was injected, and peaks were eluted with a gradient of 25–95% CH3CN/H2O (both with 0.1% formic acid) over 5 min, then 95% CH3CN/H2O for 2 min.

Synthetic protocols Ethyl 3-oxo-2-(2-oxo-1,2-dihydropyridin-1-yl)-3-phenylpropanoate (3); ethyl 3-oxo-3-phenyl-2-(pyridin-2-yloxy)propanoate (4) A suspension of 2-hydroxypyridone (163 mg, 1.72 mmol), tetrabutylammonium bromide (59.5 mg, 0.184 mmol), and potassium carbonate (765 mg, 55.3 mmol) in acetone (6.0 mL) was heated to 40 °C and stirred for 30 min. Ethyl-2-bromo-3-oxo-3-phenylpropanoate (500 mg, 1.84 mmol) was added and the suspension was stirred at 40 °C for 30 min before being cooled to 20 °C. A solution of acetic acid (160 μL, 2.79 mmol) in water (2.0 mL) was added slowly, and the mixture was stirred for 15 min. The resulting mixture was diluted with H2O (2.0 mL) and extracted with DCM (3 × 15 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated. The crude orange oil was dissolved in DCM and purified via flash chromatography (25 g SiO2 column, 0–60% EtOAc/hexanes) to yield pyridone 3 as a yellow oil (229 mg, 47%) and 4 as a yellow oil (44 mg, 9%). 3: Rf = 0.25 (50 : 50 EtOAc : hexanes); 1H NMR (CDCl3, 400 MHz) δ 8.09 (d, J = 7.3 Hz, 2H), 7.64–7.58 (m, 2H), 7.51–7.46 (m, 3H), 7.37–7.33 (m, 1H), 6.62 (d, J = 8.7 Hz, 1H), 6.22–6.18 (m, 1H), 4.31–4.28 (m, 2H), 1.27 (t, J = 7.1 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δ 191.9, 166.6, 161.4, 140.6, 136.4, 134.8, 134.0, 129.4, 129.2, 120.2, 106.4, 62.8, 59.2, 14.1; LC-MS tR = 3.96; m/z = 285.75 (M + H); 4: 1H NMR (CDCl3, 400 MHz) δ 8.11–8.05 (m, 3H), 7.63–7.58 (m, 2H), 7.51–7.46 (m, 2H), 6.98–6.92 (m, 2H), 6.68 (s, 1H), 4.27 (q, J = 7.1 Hz, 2H), 1.22 (t, J = 7.1 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δ 191.7, 166.6, 161.3, 146.5, 139.3, 133.9, 129.4, 128.7, 118.3, 111.4, 76.0, 62.2, 14.1; LC-MS tR = 2.86; m/z = 285.75 (M + H).

1-(2-Oxo-2-phenylethyl)-1,2-dihydropyridin-2-one (5) 2-Hydroxypyridine (0.263 g, 2.76 mmol) and Cs2CO3 (1.63 g, 5.02 mmol) were added to a 15 mL oven-dried flask with stir bar and sealed under N2. Anhydrous DMF (5.0 mL) was added and the suspension was stirred at 20 °C for 30 min before 2-bromoacetophenone (0.500 g, 2.51 mmol) was added and the suspension was stirred for an additional 1 h at 20 °C. A solution of glacial acetic acid (210 μL, 3.77 mmol) in H2O (2 mL) was slowly added and the mixture was stirred until it turned clear and stopped bubbling (∼10 min). The mixture was diluted with H2O (5 mL) and EtOAc (40 mL) and the layers were separated. The organic layer was washed with water (3 × 15 mL) and the combined aqueous layers were extracted with EtOAc (10 mL). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered, and concentrated. The crude yellow solid was dissolved in DCM and purified via flash chromatography (50 g SiO2 column, 0–100% EtOAc/hexanes) to yield pyridone 5 as a white crystalline solid (0.411 g, 77%). This compound has been previously reported and characterized (CAS# 952-75-0). 1H NMR (CDCl3, 400 MHz) δ 8.02–8.00 (m, 2H), 7.62 (tt, J = 7.4, 1.2 Hz, 1H), 7.51–7.47 (m, 1H), 7.40–7.35 (m, 1H), 7.23–7.21 (m, 1H), 6.59 (d, J = 9.2 Hz, 1H), 6.21 (td, J = 6.7, 1.3 Hz, 1H), 2.21 (s, 2H); 13C NMR (CDCl3, 100 MHz) δ 192.4, 162.5, 140.2, 138.4, 134.7, 134.1, 129.0, 128.2, 120.8, 106.1, 54.4.

Methyl 3-oxo-3-phenylpropanoate (10) NaH (3.7 g, 60% in mineral oil, 92 mmol) and dimethyl carbonate (5.9 g, 66 mmol) were added to a 250 mL oven-dried flask with stir bar. A reflux condenser and addition funnel were attached, the apparatus was sealed and flushed with N2, and anhydrous toluene (33 mL) was added under N2. After the mixture was heated to reflux, a solution of acetophenone (3.80 mL, 32.4 mmol) in toluene (17 mL) was added dropwise over 0.5 h. The reaction solution turned orange with the formation of a white precipitate. After the evolution of hydrogen ceased (∼15 min), the reaction was cooled to room temperature. Glacial acetic acid (10 mL) was added dropwise and a heavy pasty solid separated. Ice-cold water was slowly added until the solid dissolved completely, and the reaction mixture was diluted with EtOAc (200 mL). The organic layer was separated, washed with H2O (200 mL) and brine (200 mL), dried over anhydrous Na2SO4, filtered, and concentrated. The residue was dissolved in DCM and purified via flash chromatography (100 g SiO2 column, 0–25% EtOAc/hexanes) to yield ester 10 as an orange oil (5.65 g, 98%). This compound has been previously reported and characterized (CAS# 614-27-7).261H NMR (300 MHz, CDCl3) δ 12.51 (s, 0.22 H), 7.94 (m, 2H), 7.78 (m, 0.44 H), 7.76–7.58 (m, 0.44 H), 7.62–7.58 (m, 1 H), 7.50–7.40 (m, 2.7 H), 5.68 (s, 0.22 H), 4.01 (s, 2H), 3.80 (s, 0.65 H), 3.75 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 192.5, 173.6, 171.6, 168.0, 136.0, 133.9, 133.4, 131.4, 128.9, 128.64, 128.6, 126.2, 87.1, 52.6, 51.5, 45.8.

Benzyl 3-oxo-3-phenylpropanoate (11) A 25 mL round bottom flask was charged with methyl 3-oxo-3-phenylpropanoate (1.05 g, 5.91 mmol), benzyl alcohol (6.10 mL, 59.1 mmol), ZnO (96.0 mg, 1.18 mmol) and anhydrous toluene (5 mL). The flask was fitted with a short-path distillation head and heated in an oil bath set at 110 °C, distilling the methanol formed during the reaction. After 24 h, LC-MS analysis of the reaction mixture showed complete consumption of the starting material. The reaction mixture was filtered through a plug of celite and concentrated. The crude residue was dissolved in DCM and purified via flash chromatography (100 g SiO2 column, 0–20% EtOAc/hexanes) to give benzyl ester 11 as a light orange oil (1.43 g, 95%). This compound has been previously reported and characterized (CAS# 63888-22-2).39 This compound exists as a mixture of tautomers in CDCl3 (10 : 1 keto : enol). 1H NMR (300 MHz, CDCl3) δ 12.50 (s, enol), 7.94 (d, J = 8.0 Hz, enol), 7.90 (d, J = 8.0 Hz, 2H), 7.77 (d, J = 7.9 Hz, enol), 7.59 (t, J = 7.5 Hz, 1H), 7.48–7.28 (m, 7H), 5.73 (s, enol), 5.25 (s, enol), 5.19 (s, 2H), 4.04 (s, 2H); 13C NMR (75 MHz, CDCl3) δ 192.4, 167.5, 135.4, 133.9, 128.9, 128.7, 128.6, 128.5, 128.4, 128.3, 67.3, 46.1.

Benzyl 2-bromo-3-oxo-3-phenylpropanoate (12) In a 100 mL flask with stir bar, ester 11 (1.00 g, 3.93 mmol), N-bromosuccinimide (0.735 g, 4.13 mmol), and Amberlyst-15 (2.89 g) in ethyl acetate (30 mL) were stirred at 20 °C for 2.5 h. After completion of the reaction, as indicated by LC-MS, the reaction mixture was filtered and washed with EtOAc (2 × 20 mL). The combined organic

filtrates were dried over anhydrous Na2SO4, and concentrated. The crude product was dissolved in DCM and purified via flash chromatography (50 g SiO2 column, 0–20% EtOAc/hexanes) to yield alkyl bromide 12 as a light yellow oil (1.20 g, 92%). This compound has been previously reported and characterized (CAS# 845733-96-2).281H NMR (400 MHz, CDCl3) δ 7.94–7.92 (m, 2H), 7.58 (m, 1H), 7.43 (m, 2H), 7.30–7.23 (m, 5H) 13C NMR (100 MHz, CDCl3) δ 188.0, 165.1, 134.6, 134.3, 133.3, 129.2, 128.9, 128.6, 128.6, 128.4, 68.8, 46.4.

Benzyl 3-oxo-2-(2-oxo-1,2-dihydropyridin-1-yl)-3-phenyl-propanoate (13) A suspension of 2-hydroxypyridone (212 mg, 2.23 mmol), tetrabutylammonium bromide (65.4 mg, 0.203 mmol), and potassium carbonate (0.841 g, 6.09 mmol) in acetone (4.0 mL) was heated to 40 °C and stirred for 30 min. Then, bromide 12 (676 mg, 2.03 mmol) in acetone (0.5 mL) was added. The suspension was stirred at 40 °C for 30 min and cooled to room temperature before a solution of acetic acid (232 μL, 4.06 mmol) in water (2.0 mL) was added slowly. The resulting mixture was stirred for 15 min before it was diluted with H2O (5.0 mL) and extracted with DCM (3 × 15 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated. The crude dark orange oil was dissolved in DCM and purified via flash chromatography (50 g SiO2, 0–50% EtOAc/hexanes) to yield pyridone 13 as a colorless oil (496 mg, 70%). 1H NMR (400 MHz, CDCl3) δ 8.07–8.04 (m, 2H), 7.64 (s, 1H), 7.60 (tt, J = 7.4, 1.2 Hz, 1H), 7.48–7.44 (m, 3H), 7.35–7.26 (m, 6H), 6.61 (d, J = 9.2 Hz, 1H), 6.18 (td, J = 6.8 Hz, 1.3 Hz, 1H), 5.25 (s, 2H). 13C NMR (100 MHz, CDCl3) δ 191.7, 166.5, 161.4, 140.7, 136.4, 134.8, 134.6, 133.9, 129.4, 129.2, 128.7, 128.7, 128.4, 120.3, 106.4, 68.3, 59.4; LC-MS tR = 3.72; m/z = 348.10 (M + H).

1-(2-Hydroxy-2-phenylethyl)-1,2-dihydropyridin-2-one (14) To a 15 mL flask with stir bar, 13 (33.5 mg, 0.096 mmol) and MeOH (3.0 mL) were added. The flask headspace was flushed with N2 before 10% Pd/C (10.3, 0.0096 mmol) was added. The flask was flushed with H2 and stirred under H2 (1 atm) at 20 °C for 1 h. After 1 h, the reaction mixture was filtered through celite, condensed under vacuum and analyzed via LC-MS and 1H NMR, which indicated conversion to alcohol 14. 1H NMR of the crude product showed signals for alkyl protons at 4.36, 3.89, and 5.01 ppm, consistent with published data. This compound has been previously reported and characterized (CAS# 69914-21-2).401H NMR (400 MHz, CD3OD) δ 7.43–7.39 (m, 2H), 7.39–7.33 (m, 3H), 7.29–7.26 (m, 2H), 6.56 (d, J = 8.5 Hz, 1H), 6.30 (td, J = 6.7, 1.2 Hz, 1H), 5.01 (dd, J = 8.8, 3.8 Hz, 1H), 4.36 (dd, J = 13.1, 3.8 Hz, 1H), 3.89 (dd, J = 13.1, 8.8 Hz, 1H). 215.75; LC-MS tR = 1.68; m/z = 215.75 (M + H).

N-[2-(Diethylamino)ethyl]-1H-imidazole-1-carboxamide (16) 1,1-Carbonyldiimidazole (205 mg, 1.26 mmol) was added to a 4 mL oven-dried vial with stir bar and sealed under N2. Anhydrous THF (1.2 mL) was added and the solution was cooled to 0 °C in an ice bath. A solution of N,N-diethylethylenediamine (97.9 mg, 0.842 mmol) in anhydrous DCM (0.6 mL) was added dropwise over 10 min and the solution was stirred at 20 °C for 1.5 h. Next, the solvent was removed via vacuum and the crude product was loaded on to celite and purified via flash chromatography (12 g C18, MeOH/H2O gradient) to yield urea 16 as a yellow oil (93.7 mg, 53%). This compound has been previously reported (CAS# 698388-51-1).411H NMR (300 MHz, CDCl3) δ 8.18 (s, 1H), 7.45–7.42 (m, 1H), 7.09–7.07 (m, 2H), 3.46 (t, J = 6.1 Hz, 2H), 2.66 (t, J = 5.6 Hz, 2H), 2.61–2.51 (m, 4H), 1.06–0.97 (m, 6H); 13C NMR (75 MHz, CDCl3) δ 149.1, 136.1, 130.3, 130.1, 116.1, 51.3, 46.8, 38.2, 11.8; LC-MS tR = 1.01; m/z = 210.80 (M + H).

N-tert-Butyl-3-oxo-2-(2-oxo-1,2-dihydropyridin-1-yl)-3-phenyl propanamide (19) In a 4 mL oven-dried vial with a stir bar, t-butylisocyanate (7.0 mg, 0.071 mmol) and NaH (60% w/w in mineral oil, 4.7 mg, 0.118 mmol) were mixed in anhydrous toluene (0.5 mL) under N2 at 20 °C. Then, ketone 5 (10.0 mg, 0.047 mmol) was added in one portion and the mixture was heated at 100 °C for 2 h. After 2 h, the reaction was quenched by the addition of saturated ammonium chloride. The aqueous layer was extracted with DCM (3 × 10 mL). The combined organic extracts were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered, and

condensed to a yellow residue. The crude product was purified via flash chromatography (10 g SiO2, 0–100% EtOAc/hexanes) to afford amide 19 as a yellow oil (2.2 mg, 15%) and recovered ketone 5 (8.0 mg, 80%). 1H NMR (300 MHz, CDCl3) δ 7.98 (d, J = 8.0 Hz, 2H), 7.74–7.72 (m, 1H), 7.56–7.52 (m, 1H) 7.43–7.36 (m, 3H), 7.31 (s, 1H), 6.85 (br s, 1H), 6.61 (d, J = 9.0 Hz, 1H), 6.29 (t, J = 6.8 Hz, 1H). LC-MS tR = 2.96; m/z = 313.15 (M + H).

2,2-Difluoro-6-methoxy-4-phenyl-2H-1λ3,3,2λ4-dioxaborinine (20a) To an oven-dried 20 mL vial, 10 (0.420 g, 2.36 mmol) and a stir bar were added and the vial was purged with N2 for 10 min. Then, anhydrous toluene (10 mL) and boron trifluoride etherate (0.58 mL, 4.71 mmol) were sequentially added. The reaction mixture was stirred at 20 °C for 20 h. The reaction mixture was then concentrated to ∼1/3 of its volume and cooled to −30 °C in a dry ice MeOH/H2O cooling bath. The precipitated material was filtered off and washed with 5 : 1 petroleum ether/EtOAc (5 mL), yielding the boron complex 20a as a yellow solid (307 mg, 58%). 1H NMR (300 MHz, CDCl3) δ 7.96–7.92 (m, 2H), 7.63–7.57 (m, 1H), 7.51–7.46 (2H), 6.03 (s, 1H), 4.12 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 179.6, 176.3, 134.0, 132.0, 129.0, 127.9, 83.2, 56.1.

Ethyl 2-(2-oxo-1,2-dihydropyridin-1-yl)acetate (21) To an oven-dried 1.0 L round bottom flask charged with a stir bar, NaH (60% in mineral oil, 1.99 g, 49.9 mmol) was suspended in anhydrous DMF (100 mL) and cooled to 0 °C in an ice bath under N2. To the NaH suspension, a solution of 2-hydroxypyridine (4.07 g, 42.1 mmol) in anhydrous DMF (200 mL) was slowly added. The resulting solution was stirred for 1 h at 0 °C before ethyl bromoacetate (4.3 mL, 38 mmol) was added and the mixture was stirred at room temperature for 1.5 h. The reaction was quenched with saturated NH4Cl (40 mL) and the product was extracted with DCM (3 × 150 mL). The combined organic extracts were washed with water (7 × 200 mL), dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was dissolved in DCM and purified via flash chromatography (100 g SiO2, 0–100% EtOAc/hexanes) to yield ester 21 as a pale yellow oil (3.62 g, 53%). This compound has been previously reported and characterized (CAS# 80056-43-5).30Rf = 0.74 (DCM/MeOH 9 : 1). 1H NMR (300 MHz, CDCl3) δ 1.29 (t, 3H, J = 7.1 Hz), 4.24 (q, 2H, J = 7.1 Hz), 4.64 (s, 2H), 6.18–6.23 (m, 1H), 6.58–6.61 (m, 1H), 7.21–7.24 (m, 1H), 7.34–7.40 (m, 1H). 13C NMR (100 MHz, CDCl3) δ 14.2, 50.5, 61.9, 106.2, 121.0, 121.0, 138.0, 140.3, 162.5, 167.9.

N-[2-(Diethylamino)ethyl]-3-oxo-3-phenylpropanamide (22) N,N-Diethylethylenediamine (167.0 mg, 1.438 mmol) was added to a 15 mL oven-dried flask with stir bar and sealed under N2. Anhydrous MeCN (5.0 mL) and 20a (250 mg, 1.11 mmol) were added, and the reaction mixture was stirred at 20 °C for 4 h. After 4 h, an aliquot was removed, condensed under reduced pressure, and dissolved in CDCl3 to monitor the reaction via1H NMR. The reaction mixture was condensed under vacuum, dissolved in EtOAc (30 mL), washed with H2O (2 × 10 mL), dried over MgSO4, filtered, and condensed under vacuum to yield complex 22 as a pale yellow oil (283 mg, 82%). The crude oil (259 mg, 0.834 mmol), sodium acetate (0.342 g, 4.17 mmol), ethanol (5.0 mL), and H2O (5.0 mL) were refluxed for 8 h. TLC analysis (10% MeOH/DCM) of the reaction mixture indicated the starting material was consumed. The solvent was removed under reduced pressure, and the residue was dissolved in EtOAc (30 mL) and washed with water (2 × 10 mL). The combined aqueous layers were saturated with NaCl and extracted with 9 : 1 DCM : MeOH (5 × 10 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated. The crude oil was purified by flash chromatography (10 g SiO2, 0–10% MeOH/DCM) to yield amide 22 as a pale yellow oil (147 mg, 67%). Rf = 0.50 (9 : 1 DCM : MeOH); 1H NMR (300 MHz, CDCl3) δ 7.99 (d, J = 7.4 Hz, 2H), 7.61–7.57 (m, 1H) 7.50–7.45 (m, 2H), 3.96 (s, 2H), 3.48 (q, J = 5.5 Hz, 2H), 2.79–2.73 (m, 6H), 1.14 (t, J = 7.1 Hz, 6H); 13C NMR (300 MHz, CDCl3) δ 195.16, 178.19, 136.43, 133.82, 128.84, 128.70, 51.54, 47.03, 46.53, 36.49, 10.50; LC-MS tR = 1.06; m/z = 263.15 (M + H).

2-(2-Oxo-1,2-dihydropyridin-1-yl)acetic acid (25) Ester 21 (3.59 g, 19.8 mmol) and EtOH : H2O (1 : 1, 60 mL) were added to a 250 mL oven-dried flask with stir bar and cooled to 0 °C. A 1 N aqueous solution of LiOH (40 mL, 40 mmol) was added and the solution was stirred for 2 h at 20 °C. After 2 h, ethanol was removed under vacuum and 2 M HCl was added to the aqueous solution to reach a pH ∼ 6. Next, the solution was concentrated down to dryness and the crude solid was dissolved with DCM and purified via flash chromatography (50 g SiO2, 0–10% MeOH/DCM w/0.1% formic acid) to afford 25 as a white powder (2.04 g, 67%). This compound has been previously reported and characterized (CAS# 56546-36-2).301H NMR (400 MHz, acetone-d6) δ 7.62–7.60 (m, 1H), 7.46–7.42 (m, 1H), 6.41–6.25 (m, 1H), 6.26–6.21 (m, 1H), 4.71 (s, 2H).

[2-(Diethylamino)ethyl][(4-methoxyphenyl)methyl]amine (26b) N,N-Diethylethylenediamine (1.25 g, 10.6 mmol) was added to a 100 mL oven-dried flask with stir bar and sealed under N2. i-PrOH (25 mL) was added and the flask was cooled to 0 °C. Then, p-anisaldehyde (1.58 mL, 12.8 mmol) was added dropwise, and the reaction was slowly warmed to 20 °C and stirred for 16 h. MeOH (20 mL) was added, the solution was cooled to 0 °C, and NaBH4 (1.69 g, 44.7 mmol) was added in portions over 1 h. The solution was stirred for an additional 1 h, while warmed to 20 °C. A solution of 10% NaOH in H2O (25 mL) was added and the resulting mixture was extracted with DCM (3 × 40 mL). The combined organic layers were washed with a 10% aqueous solution of NaI (50 mL). The organic layers were dried over Na2SO4, filtered, and concentrated. The resulting crude yellow oil was dissolved in DCM (5 mL) and ether (10 mL), cooled to 0 °C, and a 4 M HCl solution in 1,4-dioxane (5.6 mL, 22.4 mmol) was added dropwise. The white precipitate was filtered and washed with DCM and diethyl ether to yield an off-white solid. This was dissolved in water (30 mL), cooled to 0 °C, and the solution was basified with NaOH (to pH ∼ 9). The resulting mixture was extracted with DCM (3 × 40 mL). The combined organics were dried over Na2SO4, filtered, and concentrated to yield amine 26b as a yellow oil (1.86 g, 74% yield). This compound has been previously reported (CAS# 65875-40-3).42Rf = 0.57 (9 : 1 DCM : MeOH); 1H NMR (300 MHz, CDCl3) δ 7.23 (d, J = 8.6 Hz, 2H), 6.85 (d, J = 8.6 Hz, 2H), 3.79 (s, 3H), 3.74 (s, 2H), 2.66 (t, J = 6.2 Hz, 2H), 2.55 (t, J = 6.2 Hz, 2H), 2.49 (q, J = 7.1 Hz, 4H), 0.99 (t, 6H); 13C NMR (75 MHz, CDCl3) δ 158.6, 132.8, 129.3, 113.8, 55.3, 53.5, 52.7, 47.1, 46.9, 11.9; LC-MS tR = 0.89; m/z = 236.90 (M + H).

N-[2-(Diethylamino)ethyl]-2-(2-oxo-1,2-dihydropyridin-1-yl)acetamide (27a) Carboxylic acid 25 (37.0 mg, 0.242 mmol) was added to an oven-dried 50 mL flask with stir bar and sealed under N2. To the suspension, anhydrous DCM (4.0 mL), HOBt (69.4 mg, 0.363 mmol), DIPEA (62.0 μL, 0.362 mmol), and N,N-diethyethylenediamine (33.7 mg, 0.290 mmol) were sequentially added. The reaction mixture was stirred for 5 min at 20 °C before EDC-HCl (69.4 mg, 0.362 mmol) was added in one portion. The reaction mixture was stirred at 20 °C for 48 h. Monitoring via LC-MS showed conversion to the desired product. The solvent was removed under vacuum and the residue was dry loaded using celite and purified via flash chromatography (12 g C18, 0–95% MeOH/H2O gradient w/0.1% NH4OH) to yield amide 27a as a pale yellow oil (21 mg, 35%). 1H NMR (300 MHz, CDCl3) δ 7.38–7.38 (m, 2H), 6.61 (d, J = 9.0 Hz, 1H), 6.24 (td, J = 1.3, 6.7 Hz, 1H), 4.56 (s, 2H), 3.30 (q, J = 5.7 Hz, 2H), 2.57–2.50 (m, 7H), 0.99 (t, J = 7.1 Hz, 6H); 13C NMR (75 MHz, CD3OD) δ 168.1, 163.6, 141.5, 139.8, 139.7, 119.3, 107.2, 52.0, 51.2, 46.9, 36.6, 10.3; LC-MS tR = 0.99; m/z = 251.80 (M + H).

N-[2-(Diethylamino)ethyl]-N-[(4-methoxyphenyl)methyl]-2-(2-oxo-1,2-dihydropyridin-1-yl)acetamide (27b) Carboxylic acid 25 (0.300 g, 19.6 mmol) was added to an oven dried 50 mL flask with stir bar and sealed under N2. Anhydrous DCE (20.0 mL) was added, and the resulting mixture was cooled to 0 °C using an ice bath before EDC-HCl (0.563, 29.4 mmol), amine 27b (0.300 g, 19.6 mmol), and DMAP (23.9 mg, 1.96 mmol) were added. The reaction was allowed to warm up to 20 °C and stirred under N2 for 16 h. The resulting solution was washed with 1 M NaOH (10 mL). The aqueous layer was extracted with DCM (2 × 5 mL), and the combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated. The crude yellow oil was dissolved with DCM and

purified via flash chromatography (12 g C18, 0–95% MeOH/H2O with 0.1% NH4OH) to yield amide 27b as a colorless oil (213 mg, 29%). The 1H and 13C NMR are complicated due to rotamers. 1H NMR (300 MHz, CDCl3) δ 7.39–7.24 (m, 3H), 7.20–7.18 (m, 1H), 6.93–6.91 (m, 1H), 6.85–6.82 (m, 1H), 6.58–6.54 (m, 1H), 6.22–6.16 (m, 1H), 4.89 (s, 1H), 4.73 (s, 1H), 4.66 (s, 1H), 4.59 (s, 1H), 3.80–3.77 (s, 3H), 3.46–3.37 (m, 2H), 2.64–2.45 (m, 6H), 1.03–0.95 (m, 6H); 13C NMR (75 MHz, CDCl3) δ 167.0, 166.7, 162.5, 162.4, 159.2, 159.0, 140.0, 140.0, 138.9, 138.7, 129.5, 129.1, 128.2, 127.9, 120.4, 114.3, 114.0, 105.8, 105.7, 55.3, 55.2, 51.5, 51.3, 50.3, 49.7, 49.4, 48.9, 47.5, 47.4, 45.6, 45.3, 45.2, 12.0; LC-MS tR = 1.43; m/z = 371.95 (M + H).

Benzyl 2-bromo-3-hydroxy-3-phenylpropanoate (35a) To a 25 mL flask with stir bar, benzyl cinnamate (477 mg, 2.00 mmol), NBS (427 mg, 2.40 mmol) and MeCN : H2O (4 : 1) (10 mL) were added and the solution was cooled to 0 °C. Iodine (50.8 mg, 0.200 mmol) was added and the mixture was stirred for 72 h. The reaction mixture was washed with 10% aq. sodium thiosulfate (20 mL) and extracted with EtOAc (3 × 40 mL). The combined organic extracts were washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was dissolved in DCM and purified by flash chromatography (50 g SiO2, 0–100% EtOAc/hexanes) to afford bromohydrin 35a as a colorless liquid (276 mg, 41%). This compound has been previously reported (CAS# 1332928-50-3).43Rf = 0.47 (50 : 50 EtOAc : hexanes); 1H NMR (300 MHz, CDCl3) δ 7.23–7.37 (m, 10H), 5.20 (s, 2H), 5.07 (dd, J = 5.5, 8.2 Hz, 1H), 4.41 (d, J = 8.2 Hz, 1H), 3.26 (br d, J = 5.5 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ 169.3, 139.0, 134.9, 128.9, 128.7, 128.7, 128.3, 127.0, 75.3, 68.0, 47.8.

Methyl 2-bromo-3-hydroxy-3-phenylpropanoate (35b) The procedure for the synthesis of bromohydrin 35a was used with the following modifications: methyl trans-cinnamate (1.62 g, 10.0 mmol) was used instead of benzyl trans-cinnamate, NBS (2.14 g, 12.0 mmol), MeCN : H2O (4 : 1) (50 mL), and iodine (254 mg, 1.00 mmol). The crude product was dissolved in DCM and purified by flash chromatography (100 g SiO2, 0–40% EtOAc/hexanes) to afford bromohydrin 35b as an off-white solid (1.45 g, 56%). This compound has been previously reported (CAS# 90841-69-3).31Rf = 0.38 (70 : 30 hexanes : EtOAc); 1H NMR (300 MHz, CDCl3) δ 7.35–7.38 (m, 5H), 5.06 (dd, J = 5.3, 8.4 Hz, 1H), 4.37 (d, J = 8.4 Hz, 1H), 3.79 (s, 3H), 3.28 (d, J = 5.3 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ 170.0, 139.0, 128.9, 128.7, 127.1, 75.3, 53.3, 47.5.

Benzyl 3-phenyloxirane-2-carboxylate (36) In a 4 mL oven-dried vial with stir bar, a suspension of 2-hydroxypryidone (6.8 mg, 0.072 mmol), tetrabutylammonium bromide (1.9 mg, 0.0060 mmol), and ground potassium carbonate (24.7 mg, 0.179 mmol) in acetone (0.5 mL) was heated at 40 °C for 30 min. A solution of the bromohydrin 35a (20.0 mg, 0.0597 mmol) in acetone (0.2 mL) was added dropwise over 5 min and the mixture was stirred at 40 °C for 30 min. The reaction mixture was monitored via LCMS and TLC (50 : 50 EtOAc : hexanes) and stained with PAA. Saturated NH4Cl (1 mL) and H2O (1 mL) were added the solution was stirred for 10 min. The resulting solution was diluted with H2O (5 mL) and extracted with DCM (3 × 10 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated. The crude pale yellow oil was dissolved in DCM and purified by flash chromatography (5 g SiO2, 0–100% EtOAc/hexanes) to yield pyridone 36 as a colorless oil (11.4 mg, 75%). This compound has been previously reported and synthesized (CAS# 144667-57-2).441H NMR (300 MHz, CDCl3) δ 3.55 (d, J = 1.7 Hz, 1H), 4.11 (d, J = 1.7 Hz, 1H), 5.25 (m, 2H), 7.25–7.40 (m, 10H); 13C NMR (75 MHz, CDCl3) δ 168.2, 135.1, 135.0, 129.2, 128.8, 128.8, 128.7, 126.0, 67.6, 58.2, 56.9.

Methyl 2-bromo-3-[(tert-butyldimethylsilyl)oxy]-3-phenylpropanoate (37a) Bromohydrin 35a (70.0 mg, 0.209 mg) was added to a 4 mL oven-dried vial with stir bar and sealed under N2. Anhydrous DCM (1.0 mL), 2.6-lutidine (92.0 μL, 0.794 mmol) and TBSOTf (72.0 μL, 0.314 mmol) were sequentially added and the solution was stirred at 20 °C for 2 h. TLC analysis (50 : 50 EtOAc : hexanes) indicated

consumption of starting material. The reaction was quenched with the slow addition of saturated NaCl (2 mL), and the mixture was stirred for 15 min. The aqueous layer was extracted with ether (2 × 10 mL), and the organic layer was washed with brine (5 mL), dried over anhydrous Na2SO4, filtered, and condensed. The crude yellow oil was dissolved in DCM and purified by flash chromatography (25 g SiO2, 0–20% EtOAc/hexanes) to yield 37a as a pale yellow oil (277 mg, 77%).45 This compound has been previously reported and synthesized (CAS# 175722-72-2). Rf = 0.68 (70 : 30 hexanes : EtOAc); 1H NMR (300 MHz, CDCl3) δ 7.31–7.38 (m, 5H), 4.98 (d, J = 9.8 Hz, 1H), 4.21 (d, J = 9.8 Hz, 1H), 3.81 (s, 3H), 0.79 (s, 9H), 0.01 (s, 3H), −0.29 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 169.6, 140.1, 128.7, 127.7, 76.7, 53.0, 49.4, 25.6, 18.0, −4.7, −3.3.

Methyl 2-bromo-3-(methoxymethoxy)-3-phenylpropanoate (37b) Bromohydrin 35a (100 mg, 0.386 mmol) was added to a 4 mL oven-dried vial with stir bar and sealed under N2. Anhydrous DCM (1.0 mL) was added and the solution was cooled to 0 °C. Then, 2,6-lutidine (67.4 μL, 0.579 mmol) was added followed by dropwise addition of methoxychloromethane (44 μL, 0.579 mmol). The solution was stirred at 0 °C for 1 h and allowed to warm up to room temperature, then stirred at room temperature for 16 h under N2. After 16 h, the reaction was diluted with EtOAc (15 mL) and washed with saturated aq. NaHCO3 (10 mL). The aqueous layer was extracted with EtOAc (2 × 10 mL), and the combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated. The crude yellow oil was dissolved in DCM and purified by flash chromatography (5 g SiO2, 0–40% EtOAc/hexanes) to yield 37b as a colorless oil (78 mg, 66%). Rf = 0.59 (70 : 30 hexanes : EtOAc); 1H NMR (300 MHz, CDCl3) δ 7.40–7.35 (m, 5H), 5.00 (d, J = 10.1 Hz, 1H), 4.54–4.45 (m, 2H), 4.33 (d, J = 10.1 Hz, 1H), 3.85 (s, 3H), 3.25 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 169.4, 136.9, 129.1, 128.7, 128.6, 128.4, 128.4, 127.1, 94.8, 78.7, 56.1, 53.1, 47.3.

Methyl 3-(acetyloxy)-2-bromo-3-phenylpropanoate (39) Bromohydrin 35b (3.28 g, 12.6 mmol) was added to a 50 mL oven-dried flask with stir bar and sealed under N2. Anhydrous DCM (20.0 mL), acetic anhydride (1.34 mL, 14.3 mmol) and DMAP (61.8 mg, 0.506 mmol) were sequentially added. The solution was stirred at 20 °C for 16 h. TLC analysis (50 : 50 hexanes : EtOAc) confirmed consumption of starting material. The reaction mixture was poured into ice cold H2O (100 mL), and extracted with DCM (3 × 100 mL). The combined organic layers were washed with brine (100 mL), dried over anhydrous Na2SO4, filtered, and concentrated. The crude pale yellow oil was dissolved in DCM and purified by flash chromatography (100 g SiO2, 0–20% EtOAc/hexanes) to yield 39 as a colorless oil (3.55 g, 93%). This compound has been previously reported and synthesized (CAS# 59339-56-9).46Rf = 0.73 (50 : 50 EtOAc : hexanes); 1H NMR (300 MHz, CDCl3) δ 7.43–7.35 (m, 5H), 6.11 (d, J = 9.9 Hz, 1H), 4.50 (d, J = 9.9 Hz, 1H), 3.81 (s, 3H), 2.02 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 168.8, 168.3, 136.0, 129.3, 128.6, 128.0, 75.6, 53.3, 46.1, 20.9.

Methyl 2-(acetyloxy)-3-(2-oxo-1,2-dihydropyridin-1-yl)-3-phenylpropanoate (40b) 2-Hydroxypyridine (3.55 g, 37.4 mmol) and Cs2CO3 (8.11 g, 24.9 mmol) were added to a 250 mL oven-dried flask with stir bar and sealed under N2. Anhydrous DMF (75 mL) was added and the suspension was heated at 50 °C for 1 h, then cooled to 20 °C. A solution of alkyl bromide 39 (7.50 g, 24.9 mmol) in anhydrous DMF (20 mL) was added and the reaction mixture was stirred at 20 °C for 16 h. The reaction was quenched with saturated aq. NH4Cl (75 mL), diluted with EtOAc (500 mL), and washed with H2O (6 × 200 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated. The resulting crude yellow oil was dissolved in DCM and purified by flash chromatography (100 g SiO2, 0–100% EtOAc/hexanes) to yield 40b as a waxy off-white solid (1.98 g, 25%) and 41b as an off-white solid (0.60 g, 9%). 40b: 1H NMR (400 MHz, CDCl3) δ 7.49–7.47 (m, 2H), 7.39–7.38 (m, 3H), 7.27–7.25 (m, 1H), 7.05 (d, J = 7.1 Hz, 1H), 6.87 (d, J = 5.1 Hz, 1H), 6.60 (d, J = 9.7 Hz, 1H), 6.02 (t, J = 6.7 Hz, 1H), 5.89 (d, J = 7.1 Hz, 1H), 3.65 (s, 3H), 2.14 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 169.4, 167.9, 162.2, 139.4, 135.9, 134.6, 129.5, 129.3, 129.1, 120.7, 105.8, 71.3, 57.2, 53.0, 20.7; LC-MS tR = 5.50; m/z = 255.75 (M + H). 41b: 1H NMR (400 MHz, CDCl3) δ 7.47–7.40 (m, 7H), 7.10 (ddd, J = 6.8, 2.1, 0.7 Hz, 1H), 6.66–6.63 (m, 2H),

6.27 (td, J = 6.8, 1.2 Hz, 1H), 3.69 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 164.0, 161.8, 151.1, 140.6, 137.5, 131.2, 129.5, 129.2, 126.6, 122.1, 115.8, 105.9, 52.0; LC-MS tR = 4.25; m/z = 255.75 (M + H).

2-Hydroxy-3-(2-oxo-1,2-dihydropyridin-1-yl)-3-phenylpropanoic acid (42b) To a 50 mL flask with stir bar, acetate 40b (1.98 g, 6.28 mmol), THF (48 mL), H2O (12 mL), and LiOH–H2O (580 mg, 13.8 mmol) were sequentially added and the reaction was stirred at 20 °C for 30 min. Analysis via LC-MS indicated complete conversion. THF was removed under vacuum before a 1 M HCl solution was added dropwise until the pH reached ∼1. The solution was concentrated, the crude product was dry loaded using celite, and purified via flash chromatography (12 g C18, 0–95% 0.5 N NH3 in MeOH/H2O) to afford carboxylic acid 42b as an off-white solid (1.49 g, 92%). 1H NMR (400 MHz, DMSO-d6) δ 7.49–7.47 (m, 3H), 7.37–7.29 (m, 4H), 6.43–6.40 (m, 2H), 6.15 (t, J = 6.7 Hz, 1H), 4.66 (d, J = 5.0 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 173.2, 161.2, 139.7, 137.6, 136.8, 129.3, 128.6, 128.1, 119.2, 105.0, 70.4, 58.2; LC-MS tR = 3.34; m/z = 258.05 (M − H).

(2-Aminoethoxy)(tert-butyl)dimethylsilane (44) Ethanolamine (2.07 g, 33.9 mmol) was added to a 25 mL oven-dried flask with stir bar and sealed under N2. Imidazole (4.61 g, 67.8 mmol) and anhydrous DCM (30 mL) were added before a solution of TBSCl (5.11 g, 33.9 mmol) in anhydrous DCM (5 mL) was added dropwise over 10 min via syringe pump at 20 °C. The solution was allowed to stir at room temperature for 1 h. Next, the solution was diluted with DCM (150 mL) and washed with water (3 × 50 mL) and brine (50 mL). The organic layer was dried over MgSO4, filtered, and concentrated to give amine 44 as a pale yellow oil (4.77 g, 80%). This compound has been previously reported and synthesized (CAS# 101711-55-1).471H NMR (CDCl3, 400 MHz) δ 3.63 (t, J = 5.2 Hz, 2H), 2.77 (t, J = 5.2 Hz, 2H), 1.47, (br s, 2H), 0.91 (s, 9H), 0.07 (s, 6H); 13C NMR (CDCl3, 100 MHz) δ 65.5, 44.5, 26.1, 18.5, −5.2.

N-{2-[(tert-Butyldimethylsilyl)oxy]ethyl}-2-hydroxy-3-(2-oxo-1,2-dihydropyridin-1-yl)-3-phenylpropanamide (46) To an oven-dried 50 mL round bottom flask, acid 42 (420 mg, 1.62 mmol), amine 44 (568 mg, 3.24), anhydrous DMF (10 mL), and anhydrous DCE (10 mL) were added. Next, NMM (356 μL, 3.24 mmol) was added via syringe and HATU (739 mg, 1.94 mmol) was added in one portion. The reaction mixture was stirred at 20 °C for 16 h under N2. After 16 h, the reaction mixture was diluted with EtOAc (200 mL) and washed with H2O (2 × 50 mL) and brine (50 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and condensed under vacuum to yield a crude yellow oil. The crude oil was purified by flash chromatography (25 g SiO2, 0–100% EtOAc/hexanes) to afford amide 46 as a colorless oil (620 mg, 91%). 1H NMR (400 MHz, CDCl3) δ 7.44–7.40 (m, 1H), 7.35–7.30 (m, 7H), 6.81 (br s, 1H), 6.68 (d, J = 9.1 Hz, 1H), 6.25 (t, J = 7.3 Hz, 1H), 6.00 (s, 1H), 4.84 (s, 1H), 3.66–3.53 (m, 2H), 3.42–3.30 (m, 2H), 0.87 (s, 9H), 0.02 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 170.9, 164.9, 140.6, 139.2, 135.0, 128.7, 128.6, 128.4, 121.6, 107.7, 74.5, 71.5, 61.8, 41.6, 26.0, 18.4, −5.3; LC-MS tR = 5.74; m/z = 417.00 (M + H).

N-{2-[(tert-Butyldimethylsilyl)oxy]ethyl}-2-oxo-3-(2-oxo-1,2-dihydropyridin-1-yl)-3-phenylpropanamide (47) Alcohol 46 (205 mg, 0.492 mmol) was added to a 20 mL oven-dried vial with stir bar and sealed under N2. Anhydrous DCM (10 mL) and DMP (251 mg, 0.592 mmol) were added and the reaction was stirred at 20 °C for 2 h under N2. A 10% Na2S2O3 aqueous solution (10 mL) was added and the biphasic mixture was stirred for 20 min until the two layers became clear. The aqueous layer was separated and the organic layer was washed with saturated NaHCO3 (2 × 5 mL). The combined aqueous layers were extracted with EtOAc (1 × 10 mL) and the combined organics were dried over anhydrous Na2SO4, filtered, and concentrated. The crude oil was dissolved in DCM and purified via flash chromatography (25 g SiO2, 0–80% EtOAc/hexanes) to afford ketone 47 as an off-white solid (170 mg, 83%). 1H NMR (400 MHz, CDCl3) δ 7.38–7.26 (m, 7H), 6.93 (d, J = 7.0 Hz, 1H), 6.85 (s, 1H), 6.52 (d, J = 9.1 Hz, 1H), 6.08 (t, J = 6.7 Hz, 1H), 3.64–3.60 (m, 2H), 3.43–3.25 (m, 2H), 0.80 (s, 9H), −0.04 (s,

6H); 13C NMR (100 MHz, CDCl3) δ 189.8, 162.4, 159.4, 140.2, 135.5, 130.7, 130.2, 130.0, 129.7, 119.7, 106.5, 64.3, 61.3, 41.5, 25.9, 18.2, −5.4; LC-MS tR = 6.09; m/z = 414.95 (M + H).

N-(2-Hydroxyethyl)-2-oxo-3-(2-oxo-1,2-dihydropyridin-1-yl)-3-phenylpropanamide (48) To a 100 mL flask charged with a stir bar, silyl ether 47 (100 mg, 0.241 mmol) and MeOH (10.0 mL) were added. Then a 2% aqueous HCl in MeOH solution (10.0 mL) was added, and the reaction was stirred for 1 h at 20 °C. The solution was concentrated, dry loaded on to celite, and purified via flash chromatography (12 g C18, 0–40% MeOH/H2O) to afford alcohol 48 as a colorless oil (56.7 mg, 78%). LC-MS tR = 3.64; m/z = 300.85 (M + H). This intermediate was used directly in the next step.

Diethyl({2-[2-oxo-3-(2-oxo-1,2-dihydropyridin-1-yl)-3-phenylpropanamido]ethyl})azanium chloride (50) Alcohol 48 (40.0 mg, 0.133 mmol) was added to a 20 mL oven-dried vial with stir bar and sealed under Ar. Anhydrous MeCN (4.0 mL), DIPEA (22.8 μL, 0.133 mmol), and MsCl (30.9 μL, 0.400 mmol) were respectively added and the solution was stirred at 20 °C for 24 h. Analysis via TLC confirmed conversion to the mesylate. Diethylamine (275 μL, 2.66 mmol) was added and the reaction was heated at 70 °C for 16 h under Ar. The reaction was concentrated, dry loaded using celite, and purified via flash chromatography (10 g C18, MeOH/H2O gradient w/0.1% formic acid) to afford the free base of amine 50 as an off-white powder (11.0 mg, 28%). Rf = 0.61 (95 : 5 DCM : MeOH); 1H NMR (400 MHz, CDCl3) δ 8.61 (s, 1H), 8.03 (s, 1H), 7.51–7.49 (m, 3H), 7.41 (ddd, J = 8.8, 6.6, 1.9 Hz, 1H), 7.37–7.35 (m, 2H), 6.90 (dd, J = 7.1, 1.7 Hz, 1H), 6.55 (d, J = 8.8 Hz, 1H), 6.15 (td, J = 6.6, 1.2 Hz, 1H), 6.07 (s, 1H), 4.73–4.70 (m, 1H), 4.26–4.22 (m, 1H), 3.86–3.58 (m, 6H), 1.36–1.24 (m, 6H). This compound had poor solubility in chloroform and MeOH. The HCl salt was formed prior to 13C NMR analysis. The product was taken up into minimal H2O and 0.5 mL of 1 M HCl was added. The solution was lyophilized to afford a yellow oil. 13C NMR (75 MHz, D2O) δ 170.4, 164.2, 155.1, 143.0, 136.5, 131.0, 131.0, 130.4, 129.9, 129.8, 118.9, 109.2, 64.8, 64.6, 49.7, 45.5, 41.9, 13.3, 10.6; LC-MS tR = 1.61; m/z = 356.40 (M + H).

Ethyl 2-(2-oxo-1,2-dihydropyridin-1-yl)-3-phenylpropanoate (51) In a 20 mL oven-dried vial with stir bar, HMDS (1.90 mL, 9.11 mmol) and anhydrous THF (9.0 mL) were added under N2. The solution was cooled to 0 °C in an ice bath before n-butyl lithium (5.43 mL of a 1.6 M solution in hexanes, 8.69 mmol) was added dropwise over 10 min. The solution was cooled to −78 °C before a solution of ester 21 (1.50 g, 8.28 mmol) in anhydrous THF (25.0 mL) was added dropwise. The solution was stirred at −78 °C for 1 h under N2 before benzylbromide (985 μL, 8.28 mmol) was added dropwise. The reaction mixture was stirred at −78 °C for 1 h, allowed to warm up to 0 °C, quenched with saturated NH4Cl (10 mL), and diluted with EtOAc (50 mL). The organic layer was separated, washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated. The crude material was dissolved in DCM and purified via flash chromatography (100 g SiO2, 0–40% EtOAc/hexanes) to afford 51 as a colorless oil (1.52 g, 68%). 1H NMR (300 MHz, CDCl3) δ 7.28–7.18 (m, 4H), 7.11–7.08 (m, 2H), 7.03 (dd, J = 6.9, 1.5 Hz, 1H), 6.50 (d, J = 9.2 Hz, 1H), 6.03 (td, J = 6.8, 1.3 Hz, 1H), 5.41 (dd, J = 9.7, 5.6 Hz, 1H), 4.21 (q, J = 7.1 Hz, 2H), 3.50 (m, 1H), 3.33 (m, 1H), 1.23 (t, J = 7.1 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 169.5, 162.2, 139.6, 136.5, 136.1, 129.1, 128.7, 127.1, 120.8, 105.6, 61.9, 61.2, 36.3, 14.1; LC-MS tR = 4.67; m/z = 271.90 (M + H).

Ethyl (2Z)-2-(2-oxo-1,2-dihydropyridin-1-yl)-3-phenylprop-2-enoate (53) The procedure for the synthesis of 51 was followed using HMDS (392 mg, 2.43 mmol), anhydrous THF (9.0 mL), n-butyl lithium (1.45 mL of a 1.6 M solution in hexanes, 2.32 mmol), 21 (400 mg, 2.21 mmol) in anhydrous THF (3.0 mL), and benzaldehyde (0.247 mL, 2.43 mmol) instead of benzylbromide. After workup, the product was dissolved in DCM and purified via flash chromatography (25 g SiO2, 0–50% EtOAc/hexanes) to afford alkene 53 as an off-white solid (272 mg, 46%). 1H NMR (400 MHz, CDCl3) δ 7.83 (s, 1H), 7.43 (ddd, J = 9.0, 6.6, 2.0 Hz, 1H), 7.33–7.26 (m, 3H), 7.20–7.18 (m, 2H), 6.96 (dd, J = 6.7, 1.8 Hz, 1H), 6.67 (d, J = 8.7 Hz, 1H), 6.18 (td, J =

6.7, 1.0 Hz, 1H), 4.31 (q, J = 7.1 Hz, 2H), 1.31 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 163.4, 162.4, 140.8, 137.7, 137.4, 131.4, 130.5, 130.2, 129.9, 128.9, 121.8, 106.8, 62.0, 14.1. LC-MS tR = 6.06; m/z = 269.80 (M + H).

(2Z)-2-(2-Oxo-1,2-dihydropyridin-1-yl)-3-phenylprop-2-enoic acid (54) To a 15 mL flask with stir bar, ester 53 (136 mg, 0.504 mmol), THF (6.0 mL), H2O (1.5 mL), and LiOH–H2O (25.4 mg, 0.604 mmol) were added and the solution was stirred at 20 °C for 12 h. THF was removed under vacuum before a 1 M HCl solution was added dropwise until the pH reached ∼1. The solution was concentrated under vacuum and the crude product was dry loaded using celite and purified via flash chromatography (12 g C18, 0–75% MeOH/H2O w/0.1% formic acid) to afford carboxylic acid 54 as an off-white solid (108 mg, 89%). 1H NMR (400 MHz, CD3OD) δ 7.94 (s, 1H), 7.65 (ddd, J = 9.1, 6.7, 2.0 Hz, 1H), 7.39–7.30 (m, 4H), 7.20–7.18 (m, 2H), 6.67 (d, J = 9.1 Hz, 1H), 6.42 (td, J = 6.7, 1.0 Hz, 1H); 13C NMR (100 MHz, CD3OD) δ 166.2, 164.8, 143.6, 140.0, 139.0, 133.1, 131.7, 131.6, 130.9, 130.1, 121.6, 109.4; LC-MS tR = 4.17; m/z = 241.75 (M + H).

Ethyl 3-hydroxy-2-(2-oxo-1,2-dihydropyridin-1-yl)-3-phenylpropanoate (55) In a 100 mL oven-dried flask with stir bar, diisopropylamine (309 μL, 2.21 mmol) and anhydrous THF (35.0 mL) were added via syringe under N2. The solution was cooled to −78 °C in a dry ice/acetone bath before n-butyl lithium (1.38 mL of a 1.6 M solution in hexanes, 2.21 mmol) was added dropwise over 10 min. The solution was allowed to stir for 30 min before a solution of 21 (400 mg, 2.21 mmol) in anhydrous THF (3.0 mL) was added dropwise over 5 min. The heterogenous solution was stirred at −78 °C for 1 h before benzaldehyde (0.247 mL, 2.43 mmol) was added, and the solution was stirred at −78 °C for 2 h. The reaction was quenched at −78 °C via the addition saturated aq. NH4Cl (15 mL) and diluted with EtOAc (75 mL). The organic layer was separated, washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated. The crude material was dissolved in DCM and purified via flash chromatography (50 g SiO2, 0–100% EtOAc/hexanes) to afford alcohol 55 as a colorless oil (307 mg, 48%). The NMR spectra are complex due to diastereomers. 1H NMR (300 MHz, CDCl3) δ 7.59 (dd, J = 6.8, 1.3 Hz, 1H), 7.26–7.15 (m, 12H), 6.80 (dd, J = 6.8, 1.5 Hz, 1H), 6.39 (d, J = 9.1 Hz, 1H), 6.28 (d, J = 9.1 Hz, 1H), 6.05 (td, J = 6.7, 1.2 Hz, 1H), 5.86 (td, J = 6.7 Hz, 1.2 Hz, 1H), 5.81 (d, J = 4.3 Hz, 1H), 5.63 (d, J = 4.3 Hz, 1H), 5.45 (d, J = 7.5 Hz, 1H), 4.74 (d, J = 7.5 Hz, 1H), 4.22–4.16 (m, 4H), 1.24–1.19 (m, 6H); 13C NMR (75 MHz, CDCl3) δ 169.4, 168.2, 162.9, 162.3, 140.2, 140.1, 139.5, 138.9, 138.5, 137.9, 128.4, 128.3, 127.8, 126.4, 125.9, 120.3, 119.7, 105.9, 105.6, 73.5, 72.1, 67.5, 63.5, 62.1, 62.1, 60.4, 21.1, 14.2, 14.1, 14.0; LC-MS tR = 4.78, 4.92; m/z = 287.80 (M + H).

Ethyl 3-[(tert-butyldimethylsilyl)oxy]-2-(2-oxo-1,2-dihydropyridin-1-yl)-3-phenylpropanoate (56) The procedure for the synthesis of 37a was used with the following modifications: 55 (153 mg, 0.533 mmol), DCM (6.0 mL), 2,6-lutidine (74.4 μL, 0.639 mmol) and TBSOTf (147 μL, 0.639 mmol) were used. The crude oil was dissolved in DCM and purified via flash chromatography (25 g SiO2, 0–50% EtOAc/hexanes) to yield 56 as a pale yellow oil (187 mg, 87%). 1H NMR (300 MHz, CDCl3) δ 8.20 (dd, J = 6.9, 1.4 Hz, 1H), 7.87 (dd, J = 6.9, 1.4 Hz, 1H), 7.56–7.38 (m, 11H), 6.62 (d, J = 9.2 Hz, 1H), 6.53 (d, J = 9.2 Hz, 1H), 6.44–6.39 (m, 1H), 6.33–6.28 (m, 1H), 6.12 (d, J = 7.2 Hz, 1H), 5.87 (d, J = 3.9 Hz, 1H), 5.44 (d, J = 7.2 Hz, 1H), 4.59–4.51 (m, 1H), 4.45–4.33 (m, 3H), 1.53 (t, J = 7.1 Hz, 3H), 1.46 (t, J = 7.1 Hz, 3H), 1.13 (s, 9H), 1.11 (s, 9H), 0.29 (s, 3H), 0.25 (s, 3H), 0.02 (s, 3H), 0.00 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 169.0, 168.5, 162.2, 161.8, 139.6, 139.5, 139.4, 139.3, 138.7, 136.6, 128.3, 128.2, 128.2, 128.2, 127.0, 126.4, 120.4, 119.7, 105.4, 104.5, 75.6, 75.4, 62.1, 61.9, 61.7, 61.5, 25.8, 25.8, 18.1, 18.1, 14.2, 14.1, −4.4, −4.6, −5.3, −5.5; LC-MS tR = 6.95; m/z = 401.95 (M + H).

Prop-2-en-1-yl 2-(2-oxo-1,2-dihydropyridin-1-yl)acetate (57) NaH (60% dispersion in mineral oil, 1.77 g, 46.3 mmol) was added to a 500 mL oven-dried flask with stir bar and sealed under N2. Anhydrous DMF (125 mL) was added and the suspension was cooled to 0 °C in an ice bath. A solution of 2-hydroxypyridine (4.00 g, 42.1 mmol) in anhydrous DMF (25.0 mL) was slowly added and stirred for 1 h at 0 °C. Allyl chloroacetate (5.98 mL, 50.5 mmol) was added and the mixture was stirred at 20 °C for 12 h.

The reaction was quenched with saturated NH4Cl (200 mL) and diluted with EtOAc (750 mL). The organic layer was washed with water (6 × 200 mL), dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was dissolved in DCM and purified via flash chromatography (100 g SiO2, 0–85% EtOAc/hexanes) to afford pyridone 57 as a pale yellow oil (3.40 g, 42%). 1H NMR (400 MHz, CDCl3) δ 7.30 (ddd, J = 9.2, 6.7, 2.1 Hz, 1H), 7.20 (ddd, J = 6.7, 2.1, 0.7 Hz, 1H), 6.49 (ddd, J = 9.2, 1.4, 0.7 Hz, 1H), 6.13 (td, J = 6.7, 1.4 Hz, 1H), 5.82 (ddt, J = 17.2, 10.5, 5.7 Hz, 1H), 5.25 (dq, J = 17.2 1.5 Hz, 1H), 5.17 (dq, J = 10.5, 1.3 Hz, 1H), 4.60 (s, 2H), 4.58 (dt, J = 5.7, 1.4 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 167.5, 162.4, 140.3, 138.0, 131.3, 121.0, 119.0, 106.2, 66.3, 50.5. LC-MS tR = 2.30; m/z = 181.90 (M + H).

Prop-2-en-1-yl 3-hydroxy-2-(2-oxo-1,2-dihydropyridin-1-yl)-3-phenylpropanoate (58) In an oven-dried 100 mL flask with stir bar, anhydrous THF (50.0 mL) and diisopropylamine (1.55 mL, 11.0 mmol) were added via syringe under N2. The solution was cooled to −78 °C before n-butyl lithium (6.90 mL of a 1.6 M solution in hexanes, 11.0 mmol) was added dropwise over 5 min. The solution was stirred for 30 min before a solution of ester 57 (1.94 g, 10.0 mmol) in anhydrous THF (8.0 mL) was added dropwise over 5 min and stirred at −78 °C for 1 h. Benzaldehyde (1.53 mL, 15.1 mmol) was added and the resulting solution was stirred at −78 °C for 2 h. The reaction was quenched at −78 °C with saturated aq. NH4Cl and diluted with EtOAc (200 mL) and H2O (10 mL). The layers were separated and the organic layer was washed with brine, dried over anhydrous Na2SO4, and concentrated. The crude yellow oil was dissolved in DCM and purified via flash chromatography (100 g SiO2, 0–70% EtOAc/hexanes) to afford alcohol 58 as a yellow oil (2.45 g, 82%). 1H NMR (400 MHz, CDCl3) δ 7.43 (d, J = 7.9 Hz, 1H), 7.27–7.22 (m, 13H), 6.71 (dd, J = 6.8, 1.5 Hz, 1H), 6.46 (d, J = 9.1 Hz, 1H), 6.40 (d, J = 8.8 Hz, 1H), 6.09 (td, J = 6.8, 1.3 Hz, 1H), 5.94–5.81 (m, 3H), 5.73 (s, 2H), 5.57 (d, J = 7.9 Hz, 1H), 5.34–5.20 (m, 4H), 4.72–4.64 (m, 6H); 13C NMR (100 MHz, CDCl3) δ 169.2, 167.9, 163.1, 162.4, 140.3, 140.2, 139.4, 138.7, 138.5, 137.9, 131.4, 131.3, 128.5, 128.4, 128.4, 128.0, 126.4, 125.9, 120.5, 120.0, 119.1, 119.0, 106.0, 105.8, 73.7, 72.1, 68.0, 66.6, 66.6, 64.0; LC-MS tR = 4.40, 4.53; m/z = 299.75 (M + H).

3-Hydroxy-2-(2-oxo-1,2-dihydropyridin-1-yl)-3-phenylpropanoic acid (59) Alcohol 58 (1.90 g, 0.264 mmol) and Pd(PPh3)4 (12.2 mg, 0.0106 mmol) were added to a 500 mL oven-dried flash with stir bar and sealed under N2. Anhydrous THF (6.0 mL) and morpholine (24.2 μL, 0.277 mmol) were added via syringe and the reaction was stirred at 20 °C for 30 min. After 30 min, analysis via LC-MS indicated consumption of starting material. The reaction mixture was concentrated, dry loaded using celite, and purified using flash chromatography (30 g C18, 0–95% MeOH/H2O w/0.1% formic acid) to afford carboxylic acid 59 as a yellow oil (1.20 g, 73%). 1H NMR (400 MHz, CD3OD) δ 8.10 (d, J = 6.5 Hz, 1H), 7.44 (d, J = 6.5 Hz, 1H), 7.36–7.15 (m, 10H), 6.37 (d, J = 9.0 Hz, 1H), 6.30–6.27 (m, 1H), 6.14–6.10 (m, 1H), 5.73 (d, J = 3.8 Hz, 1H), 5.45 (d, J = 7.9 Hz, 1H), 5.30 (d, J = 7.9 Hz, 1H). LC-MS tR = 3.74, 3.88; m/z = 259.75 (M + H).

N-[2-(Diethylamino)ethyl]-3-hydroxy-2-(2-oxo-1,2-dihydropyridin-1-yl)-3-phenylpropanamide (60) Carboxylic acid 59 (886 mg, 3.42 mmol) was added to a 500 mL oven-dried flask with stir bar and sealed under N2. Anhydrous DCM (150 mL), HATU (1.95 g, 5.13 mmol), DIPEA (655 μL, 3.76 mmol), and ,N-diethyethylenediamine (624 μL, 4.44 mmol) were sequentially added and the solution was stirred at 20 °C for 12 h. After 12 h, the reaction was concentrated under vacuum, dry loaded using celite, and purified via flash chromatography (30 g C18, 0–95% MeOH/H2O w/0.1% formic acid) to afford amide 60 as an off-white waxy solid (918 mg, 75%). m.p. 75–77 °C; 1H NMR (400 MHz, CD3OD) δ 7.44 (dd, J = 6.9, 1.5 Hz, 1H), 7.34 (ddd, J = 9.0, 6.7, 2.0 Hz, 1H), 7.28–7.26 (m, 2H), 7.24–7.19 (m, 3H), 6.36 (dd, J = 9.0, 0.6 Hz, 1H), 6.14 (td, J = 6.7, 1.3 Hz, 1H), 5.39 (d, J = 9.5 Hz, 1H), 5.19 (d, J = 9.5 Hz, 1H), 3.79–3.73 (m, 1H), 3.64–3.57 (m, 1H), 3.38–3.22 (m, 6H), 1.31 (t, J = 7.3 Hz, 6H); 13C NMR (100 MHz, CD3OD) δ 171.8, 164.4, 142.5, 141.1, 140.0, 129.5, 129.4, 128.0, 120.5, 108.4, 73.4, 67.3, 53.0, 49.1, 35.7, 9.2. LC-MS tR = 1.07, 1.32; m/z = 357.95 (M + H).

N-[2-(Diethylamino)ethyl]-3-oxo-2-(2-oxo-1,2-dihydropyridin-1-yl)-3-phenylpropanamide (61) Alcohol 60 (47.5 mg, 0.133 mmol) was added to a 20 mL oven-dried vial with stir bar and sealed under N2. Anhydrous DCM (6.0 mL) and DMP (84.5 mg, 0.199 mmol) were sequentially added and the reaction mixture was stirred at 20 °C for 1 h. After 1 h, H2O (2.7 μL, 0.15 mmol) was added via microsyringe. The reaction solution was stirred at room temperature for 48 h before being quenched with saturated Na2S2O3 (3 mL). The product was extracted with DCM (3 × 10 mL) and concentrated. The crude product was dissolved in DCM and purified via flash chromatography (12 g SiO2, 0–20% MeOH/DCM) to afford ketone 61 as an off-white solid (11.7 mg, 25%). Rf = 0.71 (80 : 20 DCM : MeOH); m.p. 124–129 °C; 1H NMR (400 MHz, CDCl3) δ 8.01 (d, J = 7.1 Hz, 2H), 7.65 (dd, J = 6.8, 1.7 Hz, 1H), 7.60–7.56 (m, 1H), 7.48–7.44 (m, 1H), 7.40–7.34 (m, 2H), 6.60 (d, J = 9.2 Hz, 1H), 6.24 (td, J = 6.8, 1.3 Hz, 1H), 3.39–3.28 (m, 2H), 2.57–2.48 (m, 6H), 0.96 (t, J = 7.2 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 192.0, 165.2, 162.0, 140.7, 137.4, 135.0, 134.3, 129.1, 128.8, 119.9, 106.2, 61.6, 51.1, 46.7, 37.4, 11.5; LC-MS tR = 1.57; m/z = 356.20 (M + H).

PDI assay protocol The PDI-catalyzed reduction of insulin was assayed by measuring the increase in turbidity as detected at an optical density of 650 nm using a Spectramax M3 (Molecular Devices, Sunnyvale, CA). Compounds were assayed in a clear, flat-bottom, 96 well plate (Nunc-Immuno™ MicroWell™, Sigma-Aldrich cat# M0661-1CS), at a final volume of 100 μL. The validation assay consisted of 400 nM of PDI in a solution containing 100 mM potassium phosphate (pH 7.4), 0.1 mM bovine insulin, 2 mM EDTA and 0.5 mM DTT (all purchased from Sigma Aldrich, St Louis, MO). Compounds were used at the concentrations indicated. The reaction was performed at 25 °C for 1 h. For calculation of the IC50, we used absorbance values recorded at 25 min, after insulin aggregation is first detectable in the vehicle control compared with the corresponding value at that time point for specimens containing the test compound. Assays were performed with n = 3, and concentration-response curves were plotted using GraphPad Prism, with error bars representing standard error of the mean (SEM).

Crystallographic data CCDC 2004020 and 2004021 (Scheme 9), 2004022 (Scheme 12), and 2004023 (Fig. 3) contain the supplementary crystallographic data for this paper.†

Author contributions Proposed synthetic routes: C. D., E. G. Synthesized and characterized compounds: E. G. Conducted the insulin reduction assay: C. S., L. L., R. F. Determined X-ray crystal structures: S. V. L. Wrote and edited the manuscript: E. G., C. D.

Funding sources C. D. and E. G. thank the Foundation for Women's Wellness for supporting this project in part. E. G. thanks Arthur J. Schmitt and Marquette University for a graduate fellowship. R. F. thanks the National Heart, Lung, and Blood Institute (R35HL135775) for support.

Notes An initial version of this manuscript was posted to the preprint server ChemRxiv.21

Conflicts of interest There are no conflicts to declare.

Acknowledgements We thank Dr Sheng Cai for assistance with LC-MS, GC-MS, and NMR instruments, Dr. Chris Kelly for a sample of Bobbitt's salt, and ChemAxon Ltd for access to chemical property and structure prediction software.

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Footnote † Electronic supplementary information (ESI) available: 1H and 13C NMR spectra; additional X-ray crystallography figures and discussion. CCDC 2004020–2004023. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/d0ob01205j